The neuropathology of Alzheimer""s disease (AD) is characterized by marked neocortical Abeta deposition and signs of oxidative stress. Metabolic signs of oxidative stress in the neocortex of AD patients, widespread oxygen radical-mediated brain damage, systemic signs of oxidative stress and the response of antioxidant systems have all been observed in AD (Martins, R. N., et al., J. Neurochem. 46:1042-1045 (1986); Smith, M. A., et al., Nature 382:120-121 (1996); Ceballos-Picot, I., et al., Free Radic. Biol. Med. 20(4):579-87 (1996); Nunomura, A., et al., J. Neurosci. 19(6):1959-64 (1999)). In fact, amelioration of oxidation injury maybe the basis for the clinical benefit of vitamin E treatment in AD subjects (Sano, M., et al., N. Engl. J. Med. 336:1216-1222 (1997)).
Axcex2 is a dimer that simultaneously binds Cu and Zn. (Huang, X., et al., J. Biol. Chem. 272:26464-26470 (1997); Atwood, C. S., et al., Journal of Biological Chemistry 273:12817-12826 (1998); Lovell, M. A., et al., J. Neurol. Sci. 158(1):47-52 (1998); Huang, X., et al., Biochemistry 38:7609-7616 (1999); Garzon-Rodriguez, W., et al., J. Biol. Chem. 272:21037-21044 (1997)). It is released from cells by oxidative stress, but its normal function and role in AD are unclear. Polymers of Abeta (Axcex2), the 4.3 kD, 39-43 amino acid peptide product of the transmembrane protein, amyloid protein precursor (APP), are the main components extracted from the neuritic and vascular amyloid deposits found in the brains of those afflicted with AD. Axcex2 deposits are usually most concentrated in regions of high neuronal cell death, and may be present in various morphologies, including amorphous deposits, plaque amyloid, and amyloid congophilic angiopathy (Masters, C. L., et al., EMBO J. 4:2757(1985); Masters, C. L., et al., Proc. Natl. Acad. Sci. USA 82: 4245 (1985)). Axcex2 deposits are decorated with inflammatory response proteins. In addition, biochemical markers of severe oxidative stress, such as peroxidation adducts, advanced glycation end-products, and protein crosslinking, are located in close proximity to the deposits.
To date, the cause of Axcex2 deposits is unknown. However, it is believed that preventing the deposit formation may be a means of treating AD since growing evidence suggests that Axcex2 deposits are intimately associated with the neuronal demise that leads to dementia in AD. More specifically, genetic studies have strongly implicated the 42 residue form of Axcex2 (Axcex21-42) in the pathogenesis of AD (Maury, C. P. J., Lab. Investig. 72:4-16 (1995); Multhaup, G., et al., Nature 325:733-736 (1987)). Axcex21-42, while a minor component of biological fluids, is highly concentrated in Axcex2 deposits. This suggests that Axcex21-42 is more pathogenic than other neurotoxic Axcex2 species. See, e.g., Kuo, Y-M., et al., J. Biol. Chem. 271:4077-81 (1996); Roher, A. E., et al., J. Biol. Chem. 271:20631-20635 (1996).
The systemic deposition of amyloid is usually associated with an inflammatory response (Pepys, M. B. and Baltz, M. L., Adv. Immunol. 34:141-212 (1983); Cohen, A. S., in Arthritis and Allied Conditions, D. J. McCarty, ed., Lea and Febiger, Philadelphia (1989), pp. 1273-1293; Kisilevsky, R., Lab. Investig. 49:381-390 (1983)). For example, serum amyloid A, one of the major acute phase reactant proteins that is elevated during inflammation, is the precursor of amyloid A protein that is deposited in various tissues during chronic inflammation, leading to secondary amyloidosis (Gorevic, P. D., et al., Ann. NY Acad. Sci.:380-393 (1982)). An involvement of inflammatory mechanisms has been suggested as contributing to plaque formation in AD (Kisilevsky, R., Mol. Neurobiol. 49:65-66 (1994)). Acute-phase proteins such as alpha I -antichymotrypsin and c-reactive protein, elements of the complement system and activated microglial and astroglial cells are consistently found in AD brains.
The mechanism underlying the formation of neurotoxic Axcex2 amyloid remains unresolved. The overexpression of Axcex2 alone cannot sufficiently explain amyloid formation, since the concentration of Axcex2 required for aggregation is not physiologically plausible. Moreover, alterations in the neurochemical environment are required for amyloid formation since the presence of Axcex21-42 is normal in biological fluids such as cerebrospinal fluid (CSF) (Shoji, M., Science 258: 126 (1992); Golde et al., Science 255(5045): 728-730 (1992); Seubert, P. et al., Nature 359: 325 (1992); Haass et al., Nature 359: 322 (1992)).
Studies into the neurochemical vulnerability of Axcex2 to form amyloid suggest altered zinc and [H+] homeostasis as the most likely explanations for amyloid formation since Axcex2 is rapidly precipitated under mildly acidic conditions in vitro (pH 3.5-6.5) (Barrow C. J. and Zagorski M. G., Science 253:179-182 (1991); Fraser, P. E., et al., Biophys. J. 60:1190-1201 (1991); Barrow, C. J., et al., J. Mol. Biol. 225:1075-1093 (1992); Burdick, D., J. Biol. Chem. 267:546-554 (1992); Zagorski, M. G. and Barrow, C. J., Biochemistry 31:5621-5631 (1992); Kirshenbaum, K. and Daggett, V., Biochemistry 34:7629-7639 (1995); Wood, S. J., et al., J. Mol. Biol. 256:870-877 (1996)), and since the presence of redox inactive Zn(II) and, to a lesser extent, redox active Cu(II) and Fe(III), markedly increases the precipitation of soluble Axcex2 (Bush, A. I., et al., J. Biol. Chem. 268:16109 (1993); Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994); Bush, A. I., et al., Science 265:1464 (1994); Bush, A. I., et al., Science 268:1921 (1995)). Zinc has an abnormal metabolism in AD and is highly concentrated in brain regions where Axcex2 aggregates.
However, the complete reversibility of Zn(II)-induced Axcex21-40 aggregation in the presence of divalent metal ion chelating agents suggests that zinc binding is a reversible, normal function of Axcex2 and implicates other neurochemical mechanisms in the formation of Axcex2 deposits. A process involving irreversible Axcex2 aggregation, such as the crosslinking of Axcex2 monomers in the formation of Axcex2 polymeric species present in amyloid plaques, is thus a more plausible explanation for the formation of neurotoxic Axcex2 deposits.
The reduction by APP of copper (II) to copper (I) may lead to irreversible Axcex2 aggregation and crosslinking. More specifically, this reaction may promote an environment that enhances the production of hydroxyl radicals, which may contribute to oxidative stress in AD (Multhaup, G., et al.,Science 271:1406-1409 (1996)). A precedent for abnormal Cu metabolism already exists in the neurodegenerative disorders of Wilson""s disease and Menkes"" syndrome and possibly in familial amyotrophic lateral sclerosis (Tanzi, R. E. et al., Nature Genetics 5:344 (1993)).
Although the fundamental pathology, genetic susceptibility and biology associated with AD are becoming clearer, a rational chemical and structural basis for developing effective drugs to prevent or cure the disease remains elusive. While the genetics of AD indicate that the metabolism of Axcex2 is intimately associated with the pathogenesis of the disease as indicated above, drugs for the treatment of AD have so far focused on xe2x80x9ccognition enhancersxe2x80x9d which do not address the underlying disease processes.
The present invention is directed to the identification of agents that can be used to decrease the neurotoxicity of Axcex2 and the formation of Axcex2 polymers, and to the use of such agents to develop methods of preventing, treating or alleviating AD and/or the symptoms of AD. More specifically, the present invention is directed to the identification of agents that could be used to treat AD.
Because the ability of Axcex2 to function as an antioxidant, i.e., to generate H2O2 from O2xe2x88x92 may, in many instances, be beneficial, the invention also relates to a method for identifying an agent to be used in the treatment and/or prevention of AD and symptoms thereof, wherein said agent is capable of interfering with the interaction of O2 and Axcex2 to generate H2O2 without interfering with the SOD-like activity of Axcex2, i.e., the ability of Axcex2 to function as an antioxidant.
Thus, the invention relates to a method for the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent inhibits oxygen-dependent hydrogen peroxide formation activity, but does not inhibit the superoxide-dependent hydrogen peroxide formation, the method comprising:
(a) adding the agent to an Axcex2-containing sample;
(b) determining whether the agent is capable of inhibiting dissolved oxygen-dependent hydrogen peroxide formation; and
(c) determining whether the agent is capable of not inhibiting the Axcex2-catalyzed superoxide-dependent hydrogen peroxide formation.
In a preferred embodiment, the method of determining whether the agent is capable of not inhibiting the superoxide-dependent hydrogen peroxide formation is conducted using pulse radiolysis or the NBT assay.
In a preferred embodiment, the determination of the ability of the agent to inhibit the Axcex2-catalyzed superoxide-dependent hydrogen peroxide formation is made by determining whether Axcex2 is capable of catalytically producing Cu(I), Fe(II) or H2O2.
The invention further relates to a method for the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent is capable of altering the production of Cu(I) by Axcex2, the method comprising:
(a) adding Cu(II) to a first Axcex2 sample;
(b) allowing the first sample to incubate for an amount of time sufficient to generate Cu(I);
(c) adding Cu(II) to a second Axcex2 sample, the second sample additionally comprising a candidate pharmacological agent;
(d) allowing the second sample to incubate for the same amount of time as the first sample;
(e) determining the amount of Cu(I) produced by the first and second samples; and
(f) comparing the amount of Cu(I) produced by the first sample to the amount of Cu(I) produced by the second sample;
whereby a difference in the amount of Cu(I) produced by the sample as compared to the second sample indicates that the candidate pharmacological agent has altered the production of Cu(I) by Axcex2.
In a preferred embodiment, the amount of Cu(I) present in the first and second samples is determined by
(a) adding a complexing agent to the first and second samples, wherein the complexing agent is capable of combining with Cu(I) to form a complex compound, wherein the complex compound has an optimal visible absorption wavelength;
(b) measuring the absorbencies of the first and second samples; and
(c) calculating the concentration of Cu(I)in the first and second samples using the absorbencies obtained in (b).
In a preferred embodiment, the method is performed in a microtiter plate, and the absorbency measurements are performed by a plate reader.
In a preferred embodiment, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Cu(I) by Axcex2.
In a preferred embodiment, the first and second Axcex2 samples are biological samples such as CSF.
The method further relates to the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent is capable of altering the production of Fe(II) by Axcex2, the method comprising:
(a) adding Fe(III) to a first Axcex2 sample;
(b) allowing the first sample to incubate for an amount of time sufficient to generate Fe(II);
(c) adding Fe(III) to a second Axcex2 sample, the second sample additionally comprising a candidate pharmacological agent;
(d) allowing the second sample to incubate for the same amount of time as the first sample;
(e) determining the amount of Fe(II) produced by the first and second samples; and
(f) comparing the amount of Fe(II) present in the first sample to the amount of Fe(II) present in the second sample;
whereby a difference in the amount of Fe(II) present in the first sample as compared to the second sample indicates that the candidate pharmacological agent has altered the production of Fe(II) by Axcex2.
In a preferred embodiment, the amount of Fe(II) present in the first and second samples is determined by
(a) adding a complexing agent to the first and second samples, wherein the complexing agent is capable of combining with Fe(II) to form a complex compound, wherein the complex compound has an optimal visible absorption wavelength;
(b) measuring the absorbencies of the first and second samples; and
(c) calculating the concentration of Fe(II) in the first and second samples using the absorbencies obtained in (b).
In a preferred embodiment, the method is performed in a microtiter plate, and the absorbency measurements are performed by a plate reader.
In a preferred embodiment, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Fe(II) by Axcex2.
In a preferred embodiment, the first and second Axcex2 samples are biological samples such as CSF.
The invention further relates to a method for the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent is capable of altering the production of H2O2 by Axcex2, the method comprising:
(a) adding Cu(II) or Fe(III) to a first Axcex2 sample;
(b) allowing the first sample to incubate for an amount of time sufficient to generate H2O2;
(c) adding Cu(II) or Fe(III) to a second AD sample, the second sample additionally comprising a candidate pharmacological agent;
(d) allowing the second sample to incubate for the same amount of time as the first sample;
(e) determining the amount of H2O2 produced by the first and second samples; and
(f) comparing the amount of H2O2 present in the first sample to the amount of H2O2 present in the second sample;
whereby a difference in the amount of H2O2 present in the first sample as compared to the second sample indicates that the candidate pharmacological agent has altered the production of H2O2 by Axcex2.
In a preferred embodiment, the Axcex2 samples of (a) and (b) are a biological fluid such as CSF.
In a preferred embodiment, the determination of the amount of H2O2 present in the first and second samples is determined by
(a) adding catalase to a first aliquot of the first sample in an amount sufficient to break down all of the H2O2 generated by the sample;
(b) adding TCEP, in an amount sufficient to capture all of the H2O2 generated by the samples, to
(i) a first aliquot of the first sample;
(ii) a second aliquot of the first sample; and
(iii) the second sample;
(c) incubating the samples obtained in (b) for an amount of time sufficient to allow the TCEP to capture all of the H2O2;
(d) adding DTNB to the samples obtained in (c);
(e) incubating the samples obtained in (d) for an amount of time sufficient to generate TMB;
(f) measuring the absorbencies at 412 nm of the samples obtained in (e); and
(g) calculating the concentration of H2O2 in the first and second samples using the absorbencies obtained in (f).
In a preferred embodiment, the method is performed in a microtiter plate, and the absorbency measurements are performed by a plate reader.
In a preferred embodiment, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of H2O2 by Axcex2.
The invention further relates to a method for the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent is capable of reducing the toxicity of Axcex2, the method comprising:
(a) adding Axcex2 to a first cell culture;
(b) adding Axcex2 to a second cell culture, the second cell culture additionally containing a candidate pharmacological agent;
(c) determining the level of neurotoxicity of Axcex2 in the first and second samples; and
(d) comparing the level of neurotoxicity of Axcex2 in the first and second samples,
whereby a lower neurotoxicity level in the second sample as compared to the first sample indicates that the candidate pharmacological agent has reduced the neurotoxicity of Axcex2, and is thereby capable of being used to treat and/or prevent AD and/or symptoms thereof.
In a preferred embodiment, neurotoxicity of Axcex2 is determined by using an MTT assay, an LDH release assay or a Live/Dead assay, e.g., Live/Dead EukoLight Viability/Cytotoxicity Assay, commercially available from Molecular Probes, Inc. (Eugene, Oreg.).
In a preferred embodiment, the cells are rat cancer cells or rat primary frontal neuronal cells.
The invention further relates to a kit for determining whether an agent is capable of altering the production of Cu(I) by Axcex2 which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein
(a) the first container means contains a peptide comprising Axcex2 peptide;
(b) a second container means contains a Cu(II) salt; and
(c) a third container means contains BC anion.
In a preferred embodiment, the Axcex2 peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration from about 10 xcexcM to about 25 xcexcM.
The invention further relates to a kit for determining whether an agent is capable of altering the production of Fe(II) by Axcex2 which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein
(a) the first container means contains a peptide comprising Axcex2 peptide;
(b) a second container means contains an Fe(III) salt; and
(c) a third container means contains BP anion.
In a preferred embodiment, the Axcex2 peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration from about 10 xcexcM to about 25 xcexcM.
The invention further relates to a kit for determining whether an agent is capable of altering the production of H2O2 by Axcex2 which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein
(a) the first container means contains a peptide comprising Axcex2 peptide;
(b) a second container means contains a Cu(II) salt;
(c) a third container means contains TCEP; and
(d) a fourth container means contains DTNB.
In a preferred embodiment, the Axcex2 peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration from about 10 xcexcM to about 25 xcexcM.
The invention further relates to a method for the identification of an agent to be used in the treatment and/or prevention of AD and/or symptoms thereof, wherein the agent is capable of inhibiting redox-reactive metal-mediated crosslinking Axcex2, the method comprising:
(a) adding a redox-reactive metal to a first Axcex2 sample;
(b) allowing the first sample to incubate for an amount of time sufficient to allow Axcex2 crosslinking;
(c) adding the redox-reactive metal to a second Axcex2 sample, the second sample additionally comprising a candidate pharmacological agent;
(d) allowing the second sample to incubate for the same amount of time as the first sample;
(e) removing an aliquot from each of the first and second samples; and
(f) determining presence or absence of crosslinking in the first and second samples,
whereby an absence of Axcex2 crosslinking in the second sample as compared to the first sample indicates that the candidate pharmacological agent has inhibited Axcex2 crosslinking.
In a preferred embodiment, at (f), a western blot analysis is performed to determine the presence or absence of crosslinking in the first and second samples.
The invention further relates to a method of treating AD and/or symptoms thereof, comprising administering to a patient in need thereof an effective amount of an agent identified by any one or combination of the screening assays described above.